The overall goal of our research program is to determine the effects of environmental estrogenic chemicals on patterns and gender specificity of DNA methylation signatures in primordial germ cells (PGCs) that pass to subsequent generations and whether changes are associated with specific phenotypic abnormalities. All PGCs undergo epigenetic reprogramming at the time of sex determination. Following global erasure of DNA methylation patterns, a de novo methylation starts allowing a controlled gene expression pattern in germ cells in a gender specific manner. This window of germ cell reprogramming has been found to be susceptible to environmental chemical insult. Our hypothesis is that: 1) environmental chemicals establish DNA methylation marks in the PGC genome that are resistant to global DNA demethylation processes; 2) these chemically induced marks are mitotically and meiotically stable, and as such are maintained in eggs and sperm; and 3) specific alterations in DNA methylation patterns are transmitted to subsequent generations through the germ line and are detectable in every cell that develops from the zygote. The proposed study will focus on an estrogenic chemical bisphenol A (BPA) to which humans and wildlife are exposed. The ability of BPA to induce adverse health effects has been reported in various animal models, including transgenerational adverse outcomes, such as reduced social interaction in mice and heart disorders in zebrafish. We have shown in medaka fish that F0 embryo exposure to BPA causes transgenerational abnormalities: reduced fertilization at F2 and F3 generation and reduced embryo survival at F3 and F4 generation. The proposed studies are aimed at identifying BPA-induced epigenetic signatures in germline cells leading to transgenerational inheritance of phenotypes at environmentally relevant doses with the medaka fish model (Oryzias latipes). We have two specific aims. Specific Aim 1 will test the hypothesis that BPA establishes unique DNA methylation signatures in primordial germ cells of males and/or females that are resistant to reprogramming. Specific Aim 2 will test the hypothesis that BPA-induced DNA methylation signatures are retained in both egg and sperm and bypass reprogramming of PGCs in the F1 (directly exposed in the F0 embryos), F2, and F3 (not directly exposed) generations. This aim will focus on events during germline transgenerational transmission of epigenetic marks from adults to the subsequent generation (F2 and F3). Combined, these two specific aims should reveal unique BPA-induced epigenetic marks in the primordial germ cells in F0 and then F1 ? F3 offspring. Results from this proposed R21 study will be used to develop a R01 project directed toward identification of additional epigenetic control mechanisms, such as histone modifications and microRNAs, underlying environmental estrogen-induced transgenerational disease phenotype and their corresponding epigenetic biomarkers across several generations beyond F3 in both medaka and mice. This program will ultimately bring new insights into epigenetic mechanisms underlying transgenerational diseases in humans.